Advances and Utility of the Human Plasma Proteome

Journal of Proteome Research, Год журнала: 2021, Номер 20(12), С. 5241 - 5263

Опубликована: Окт. 21, 2021

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need understand effects COVID-19, proteomic analysis blood-derived serum plasma has become even more important for studying human biology pathophysiology. Here we provide views perspectives about developments possible clinical applications that use mass-spectrometry(MS)- affinity-based methods. We discuss examples where proteomics contributed valuable insights into SARS-CoV-2 infections, aging, hemostasis offered by combining with genetic data. As a contribution Human Proteome Organization (HUPO) Plasma Project (HPPP), present PeptideAtlas build 2021-07 comprises 4395 canonical 1482 additional nonredundant detected 240 MS-based experiments. In addition, report new Extracellular Vesicle 2021-06, which five studies 2757 extracellular vesicles blood, 74% (2047) are common PeptideAtlas. Our overview summarizes advances, impactful applications, ongoing challenges translating utility precision medicine.

Язык: Английский

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SARS-CoV-2 S2–targeted vaccination elicits broadly neutralizing antibodies

Science Translational Medicine, Год журнала: 2022, Номер 14(655)

Опубликована: Июль 27, 2022

Several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current disease 2019 (COVID-19) pandemic. Although antibody cross-reactivity with spike glycoproteins (S) diverse coronaviruses, including endemic common cold coronaviruses (HCoVs), has been documented, it remains unclear whether such responses, typically targeting conserved S2 subunit, contribute to protection when induced by infection or through vaccination. Using a mouse model, we found that prior HCoV-OC43 S–targeted immunity primes neutralizing responses otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting responses. Moreover, vaccination elicited antibodies in mice neutralized animal human alphacoronaviruses betacoronaviruses vitro provided degree against challenge vivo. Last, history Wuhan–based vaccination, further broader response than booster Wuhan suggesting may prevent repertoire focusing caused repeated homologous These data establish protective value an vaccine support notion better prepare immune system respond changing nature S1 subunit concern, as well future zoonoses.

Язык: Английский

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Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage

The Lancet Microbe, Год журнала: 2024, Номер 5(4), С. e335 - e344

Опубликована: Март 11, 2024

Язык: Английский

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Structure and inhibition of SARS-CoV-2 spike refolding in membranes

Science, Год журнала: 2024, Номер 385(6710), С. 757 - 765

Опубликована: Авг. 15, 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds the receptor angiotensin converting enzyme (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas S1 domain contains high sequence variability, is conserved a promising pan-betacoronavirus vaccine target. We applied cryo-electron tomography to capture intermediates understand inhibition by antibodies stem-helix. Subtomogram averaging revealed ACE2 dimers cross-linking spikes before transitioning into intermediates, which were captured at various stages refolding. Pan-betacoronavirus neutralizing targeting stem-helix bound inhibited prehairpin intermediates. Combined with molecular dynamics simulations, these structures elucidate process SARS-CoV-2 entry reveal how S2-targeting neutralize infectivity arresting

Язык: Английский

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SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies

eLife, Год журнала: 2022, Номер 11

Опубликована: Май 19, 2022

The outcome of infection is dependent on the ability viruses to manipulate infected cell evade immunity, and immune response overcome this evasion. Understanding process key understanding pathogenesis, genetic risk factors, both natural vaccine-induced immunity. SARS-CoV-2 antagonises innate interferon response, but whether it manipulates cellular immunity unclear. An unbiased proteomic analysis determined how surface protein expression altered SARS-CoV-2-infected lung epithelial cells, showing downregulation activating NK ligands B7-H6, MICA, ULBP2, Nectin1, with minimal effects MHC-I. This occurred at level synthesis, could be mediated by Nsp1 Nsp14, correlated a reduction in activation. identifies novel mechanism which host-shutoff Later disease process, strong antibody-dependent activation (ADNKA) developed. These responses were sustained for least 6 months most patients, led high levels pro-inflammatory cytokine production. Depletion spike-specific antibodies confirmed their dominant role neutralisation, these played only minor ADNKA compared other proteins, including ORF3a, Membrane, Nucleocapsid. In contrast, induced following vaccination was focussed solely spike, weaker than infection, not boosted second dose. insights have important implications progression, vaccine efficacy, design.

Язык: Английский

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NeutrobodyPlex—monitoring SARS‐CoV‐2 neutralizing immune responses using nanobodies

EMBO Reports, Год журнала: 2021, Номер 22(5)

Опубликована: Апрель 27, 2021

Article27 April 2021Open Access Transparent process NeutrobodyPlex—monitoring SARS-CoV-2 neutralizing immune responses using nanobodies Teresa R Wagner orcid.org/0000-0002-1050-5948 Pharmaceutical Biotechnology, Eberhard Karls University, Tuebingen, Germany Natural and Medical Sciences Institute, University of Reutlingen, Search for more papers by this author Elena Ostertag Interfaculty Institute Biochemistry, Philipp D Kaiser Marius Gramlich Natalia Ruetalo Virology Epidemiology Viral Diseases, Hospital Daniel Junker Julia Haering Bjoern Traenkle Matthias Becker Alex Dulovic Helen Schweizer orcid.org/0000-0001-9407-944X Livestock Center the Faculty Veterinary Medicine, Ludwig Maximilians Oberschleissheim, Stefan Nueske Armin Scholz Anne Zeck Katja Schenke-Layland Cluster Excellence iFIT (EXC2180) "Image-Guided Functionally Instructed Tumor Therapies", Department Women's Health, Research Medicine/Cardiology, Cardiovascular Laboratories, David Geffen School Medicine at UCLA, Los Angeles, CA, USA Annika Nelde Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Internal Cell Biology, Monika Strengert Epidemiology, Helmholtz Centre Infection Research, Braunschweig, TWINCORE GmbH, Experimental A Joint venture Hannover Hannover, Juliane S Walz Dr. Margarete Fischer-Bosch Pharmacology Robert Bosch Disease, RBCT, Stuttgart, Georg Zocher Thilo Stehle Vanderbilt Nashville, TN, Michael Schindler Nicole Schneiderhan-Marra Ulrich Rothbauer Corresponding Author [email protected] orcid.org/0000-0001-5923-8986 Information Wagner1,2,†, Ostertag3,†, Kaiser2, Gramlich2, Ruetalo4, Junker2, Haering2, Traenkle2, Becker2, Dulovic2, Schweizer5, Nueske5, Scholz5, Zeck2, Schenke-Layland2,6,7,8, Nelde6,9,10, Strengert11,12, Walz6,9,10,13, Zocher3, Stehle3,14, Schindler4, Schneiderhan-Marra2 *,1,2,6 1Pharmaceutical 2Natural 3Interfaculty 4Institute 5Livestock 6Cluster 7Department 8Department 9Clinical 10Institute 11Department 12TWINCORE 13Dr. 14Vanderbilt †These authors contributed equally to work *Corresponding author. Tel: +49 7121 51530-415; Fax: 51530-816; E-mail: EMBO Reports (2021)22:e52325https://doi.org/10.15252/embr.202052325 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In light COVID-19 pandemic, there is an ongoing need diagnostic tools monitor status large patient cohorts effectiveness vaccination campaigns. Here, we present 11 unique (Nbs) specific spike receptor-binding domain (RBD), which 8 Nbs potently inhibit interaction RBD with angiotensin-converting enzyme 2 (ACE2) as major viral docking site. Following detailed epitope mapping structural analysis, select two inhibitory Nbs, one binds inside outside RBD:ACE2 interface. Based on these, generate biparatopic nanobody (bipNb) neutralization efficacy in picomolar range. Using bipNb surrogate, establish competitive multiplex binding assay ("NeutrobodyPlex") analysis presence performance RBD-binding antibodies serum convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening infected individuals, guide vaccine design. SYNOPSIS Nanobodies its ACE2 are generated. interface form serological antibody testing. presented detection samples. allows classification individuals' potency. This study describes generation surrogates monitoring individuals. analyses Introduction As March 2021, pandemic has led deaths than 2.8 million people worldwide continues cause severe lockdowns dramatic economic losses. The emergence spread new mutants pose additional risk current campaigns (Wang et al, 2021). acute respiratory syndrome coronavirus (SARS-CoV-2) causative agent disease expresses surface glycoprotein (spike), consists subunits S1 S2 (Wrapp 2020b; Zhu 2020). For infection, located within interacts expressed human epithelial cells tract (Tai 2020; Wrapp Yan In-depth revealed spike-specific (NAbs) were shown uptake various mechanisms (Rogers Tortorici context, constantly growing number NAbs specifically targeting have been described, underlining importance blocking site development protective response (Brouwer Cao Ju Robbiani Shi Tai Yu To date, numerous preclinical clinical prophylactic therapeutic options, providing immediate protection against infection (reviewed Jiang Zohar Alter, Promising alternatives conventional (IgGs) single-domain (nanobodies, Nbs) derived from heavy-chain camelids (Fig 1) (Muyldermans, 2013). Due their small size compact fold, show high chemical stability, solubility, fast tissue penetration. can be efficiently selected different epitopes same antigen converted into multivalent formats potential address impressively demonstrated recent identification several RBD-specific naïve/ synthetic libraries (Chi 2020a; Custodio Huo preprint: Schoof Walter Ahmad 2021) immunized animals Esparza Gai Hanke Nieto Xiang Koenig Some identified potency proposed (Custodio 2021), activation fusion machinery (Koenig induction inactive conformation (preprint: Figure 1. Generation genetically engineered only alpacas. between homotrimeric protein (ACE) blocked (RBD)-specific monovalent format. Download figure PowerPoint Since outbreak, multiple assays established seroconversion individuals estimate level endemic general population. However, most available tests measure full therefore not differentiate total (Amanat Lassaunière Roxhed Stadlbauer Tang Detection latter still mostly performed virus (VNTs), both time consuming (2–4 days) require infectious virions specialized biosafety 3 (BSL3) facility (Muruato Scholer overcome these limitations, aimed employ developed approach screen basis samples patients describe selection alpaca glycosylated RBD. Employing vitro assay, effectively block RBD, S1, (spike) neutralize cell line. RBD:Nb complexes, potent simultaneously generated Nb (bipNb). represents surrogate IC50 values low range exhibits substantially improved affinities. Notably, addressing least conserved interface, capable bind recently described strains B.1.1.7 (UK) B.1.351 (South Africa). samples, implemented termed NeutrobodyPlex. data presented, provides versatile helping populations, determine success Results Selection SARS-CoV-2-specific directed SARS-CoV-2, (Vicugna pacos) purified 2020) phagemid library comprising ~ 4 × 107 clones representing repertoire variable heavy chains (VHHs Nbs). After rounds phage display passively adsorbed biotinylated immobilized streptavidin plates, analyzed 492 individual solid-phase ELISA 325 positive binders. Sequence 72 cluster eight families highly diverse complementarity determining regions (CDR) 2A, Appendix Table S1). Individual produced Escherichia coli (E. coli) 2B), affinity measurements KD ranging 1.4 53 nM indicating 10 high-affinity NM1225, displayed micromolar range, was considered further 2C, Fig Next, whether ACE2. end, utilized competition employing respective antigens coupled paramagnetic beads (MagPlex Microspheres) (Becker incubated them dilutions before measuring residual via streptavidin-PE conjugate. controls, included non-specific (GFP-Nb, negative control) inhibiting mouse (Gorshkov controls. Data obtained through showed out all tested EV1). NM1228 (0.5 nM), NM1226 (0.82 nM) NM1230 (2.12 comparable measured IgGs (MM43: 0.38 nM; MM57: 3.22 nM). 2. Biochemical characterization Amino acid sequences region after biopanning listed (upper panel). Phylogenetic tree based ClustalW alignment (lower Recombinant expression purification metal chromatography (IMAC) exclusion (SEC). Coomassie staining µg shown. biolayer interferometry (BLI)-based measurements, biosensors. Kinetic four concentrations 15.6 µM. example, sensogram indicated table summarizes affinities (KD), association (kon), dissociation constants (koff) determined Click here expand figure. EV1. ACE2-competing three spike-derived antigens: S1-domain (S1) (Spike). diluted 2.1 µM 0.12 antigen-bound measured. MFI signals normalized maximum detectable signal per given ACE2-only control. calculated four-parametric sigmoidal model. mean ± s.d. technical replicates. set diversity according CDR3 highest examined infection. test (VNT), Caco-2 co-incubated SARS-CoV-2-mNG clone serial NM1223, NM1224, NM1226, NM1228, GFP-Nb 48 h post-infection automated fluorescence-microscopy fixed nuclear-stained cells. rate, non-treated control plotted inhibition curve fits. strong 15 7 followed (~ 37 NM1224 256 (Appendix S2). expected our biochemical NM1223 found Overall, findings consistent results thus demonstrating potencies ACE2-blocking Epitope identify relative location firstly binning experiments (BLI). this, sensors, pre-coated loaded first short step subsequent loading second S3A). expected, displaying similar (NM1221, NM1222, NM1230, Nb-Set2) unable they recognize identical epitopes. Interestingly, noticed CDR3s such NM1227, NM1229 could simultaneously, suggesting also overlapping Consequently, clustered Nb-Set1. total, five distinct Nbs-Sets, candidate compared any member Nb-Set S3B). mapping, Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Nb-Sets relation previously sites EV2A, S4) (Lan Both members Nb-Set1, interacted back/ lower right (Back View, EV2B C). exchange amino (aa) NRBD370 – LRBD387 EV2B). covered YRBD489 - SRBD514, being part EV2C). (Nb-Set2) shows CRBD432 LRBD452 covering acids involved (GRBD446, YRBD449). protected NRBD487 GRBD496, overlaps EV2D). accordance studies, differ considerably Nb-Sets. NM1221 NM1222 (both addressed EV2E F) while (Nb-Set4) other side EV2G, Front View) residues upper left corner). non-inhibitory (Nb-Set3) contact but rather opposite EV2H, View). EV2. HDX mass spectrometry Surface structure model showing HDX-MS upon highlighted colors strength protection. Accordingly, residues, recognized epitopes, sequence. Numbered (pdb code: 6M17 (Yan 2020)) red. (Nb-Set1) (Nb-S

Язык: Английский

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Structure of SARS-CoV-2 M protein in lipid nanodiscs

eLife, Год журнала: 2022, Номер 11

Опубликована: Окт. 20, 2022

SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other through physical interactions directing them to sites of budding. As the most abundant protein a target patient antibodies, is compelling for vaccines therapeutics. Still, structure molecular basis its virion formation are unknown. Here, we present cryo-EM lipid nanodiscs 3.5 Å resolution. forms 50 kDa homodimer that structurally related ORF3a viroporin, suggesting shared ancestral origin. Structural comparisons reveal how intersubunit gaps create small, enclosed pocket large open cavity ORF3a, consistent with ion channel activity, respectively. displays strikingly electropositive cytosolic surface may be important N, S, RNA. Molecular dynamics simulations show high degree rigidity simple bilayer support homodimers scaffolding assembly. Together, these results provide insight roles coronavirus structure.

Язык: Английский

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Peptide microarrays coupled to machine learning reveal individual epitopes from human antibody responses with neutralizing capabilities against SARS-CoV-2

Emerging Microbes & Infections, Год журнала: 2022, Номер 11(1), С. 1037 - 1048

Опубликована: Март 23, 2022

The coronavirus SARS-CoV-2 is the causative agent for disease COVID-19. To capture IgA, IgG, and IgM antibody response of patients infected with at individual epitope resolution, we constructed planar microarrays 648 overlapping peptides that cover four major structural proteins S(pike), N(ucleocapsid), M(embrane), E(nvelope). arrays were incubated sera 67 positive 22 negative control samples. Specific responses to detectable, nine associated a more severe course disease. A random forest model disclosed binding 21 peptides, mostly localized in S protein, was higher neutralization values cellular anti-SARS-CoV-2 assays. For antibodies addressing N-terminus M, or close fusion region S, protective effects proven by depletion study pinpoints unusual viral epitopes might be suited as vaccine candidates.

Язык: Английский

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Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes

Proceedings of the National Academy of Sciences, Год журнала: 2022, Номер 119(31)

Опубликована: Июль 15, 2022

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their immediately useful. Laboratory-based genetic engineering methods enhance affinity, termed maturation, deliver useful reagents different areas of biology and potentially medicine. Using the receptor binding domain (RBD) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein a library, we generated closely related nanobodies with micromolar nanomolar affinities. By analyzing structure–activity relationship using X-ray crystallography, cryoelectron microscopy, biophysical methods, observed that higher conformational entropy losses in formation protein–nanobody complex are associated tighter binding. To investigate this, structural ensembles complexes electron microscopy maps correlated fluctuations affinity. This insight guided nanobody improved affinity protein.

Язык: Английский

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Glycosylation in SARS-CoV-2 variants: A path to infection and recovery

Biochemical Pharmacology, Год журнала: 2022, Номер 206, С. 115335 - 115335

Опубликована: Окт. 31, 2022

Язык: Английский

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